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Development 139 (23): 4405-4415

Dishevelled limits Notch signalling through inhibition of CSL

Giovanna M. Collu1,2,{ddagger}, Ana Hidalgo-Sastre1, Ahmet Acar1,*, Laura Bayston1,*, Clara Gildea1,*, Michael K. Leverentz1,*, Christopher G. Mills1,*, Thomas W. Owens1,*, Olivier Meurette3, Karel Dorey4,{ddagger}, and Keith Brennan1,{ddagger}

1 Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK
2 Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1020, New York, NY 10029, USA
3 Apoptosis, Cancer and Development Laboratory – Equipe labellisée ‘La Ligue’, Centre de Recherche en Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, 69008 Lyon, France
4 The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK


Figure 1
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  		Fig. 1. Wnt signalling inhibits Notch activity via Dishevelled. (A,B) Wnt inhibits endogenous Notch signalling. Control or Wnt1-expressing SHEP-RBPJ{kappa}-Luc cells were cultured on immobilised JAG1-Fc and Notch signalling was analysed by luciferase assays or qRT-PCR for the endogenous target Hes1. Data are presented as mean fold change (±s.e.m.) in relative luciferase units (RLU) (A) or HES1 expression normalised to HPRT1 (B), relative to control cells. Notch-induced luciferase and HES1 expression were significantly lower in Wnt1-expressing cells (two-tailed t-test, n=3). (C-E) Wnt signalling limits Notch-dependent transcriptional activity at the level of Dishevelled. CHO-K1 (C,D) and NIH3T3 (E) cells were transfected with the RBPJ{kappa}-reporter construct (RBPJ{kappa}-Luc) and a Renilla luciferase control construct (pRL-CMV). Notch signalling was activated by {Delta}N-mN1 expression. Data are presented as mean fold change (±s.e.m.) in RLU compared with {Delta}N-mN1 alone. (C) Wnt signalling was activated by expressing mWnt1, mDvl2 and S45F mβ-catenin (β-cat). Wnt1 and Dvl2 significantly inhibited Notch activity, whereas β-catenin did not (one-way ANOVA and Tukey's post-hoc tests, n=3). (D) Cells were treated with 20 mM LiCl or 10 μM SB216763 (SB) to inhibit GSK3β. Untreated cells (–) and control treatments of 20 mM KCl and DMSO are shown. None of the treatments significantly inhibited Notch activity (one-way ANOVA and Tukey's post-hoc tests, n=3). (E) Wnt1 was expressed in cells that had been previously transfected with an siRNA construct that recognises mDvl2. Dishevelled was required for Wnt-induced inhibition of Notch signalling (two-tailed t-test, n=3) (ns, P>0.05; *P<0.05; ***P<0.001).

 

Figure 2
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  		Fig. 2. Dishevelled regulates Notch-dependent cell fate decisions in vivo. (A-C) XDvl2 is required to regulate Notch signalling during development. Xenopus tropicalis embryos were injected at the one-cell stage with control morpholino (MOC) or one targeting Xdvl2 (MODvl2). Ciliated cell precursors were detected by α-tubulin expression (purple staining). (D) Precursors were counted within a box of standard area drawn over the centre of each embryo (see inset). Data are presented as mean number of precursors counted (±s.e.m.). Uninjected (UI) and MOC embryos were indistinguishable (A,B), whereas MODvl2 embryos exhibited a significant reduction in precursor number (C) (one-way ANOVA with Tukey's post-hoc tests). (E) qRT-PCR analysis of α-tubulin and esr1 expression in MOC and MODvl2 embryos. Expression was normalised to rpl8. Data are presented as mean fold change (±s.e.m.) in normalised expression values relative to uninjected embryos. esr1 expression was significantly increased in the MODvl2 embryos (two-tailed t-test, n=3). (F-I') XDvl2 expression inhibits endogenous Notch signalling and rescues the NICD gain-of-function phenotype. Xenopus laevis embryos were injected in one blastomere of the two-cell embryo with mRNA encoding β-gal (F,F'), β-gal and XDvl2 (G,G'), β-gal and XNICD (H,H'), or β-gal, XNICD and XDvl2 (I,I'). (J) Ciliated cell precursors were quantified as above (see D). X-Gal staining was performed to distinguish the injected side (pale red). Images of the uninjected and injected sides of the same embryo are shown. XDvl2 promoted (G') and XNICD inhibited (H') ciliated cell precursor specification (two-tailed paired t-test). XDvl2 completely rescued the XNICD phenotype (I') and there was no significant difference between the uninjected sides of any condition, and the β-gal or XNICD + XDvl2 expressing sides (two-way ANOVA and Bonferroni's post-hoc test). (K) qRT-PCR analysis of α-tubulin and esr1 expression in embryos expressing GFP or XDvl2 conducted as above (see E). esr1 expression was significantly decreased in XDvl2-expressing embryos (two-tailed t-test, n=3). Scale bars: 500 μm in A-C,F-I'. **P<0.01; ***P<0.001; ns, P>0.05.

 

Figure 3
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  		Fig. 3. Dishevelled does not inhibit Notch cleavage or the nuclear translocation of NICD. (A) mDvl2 inhibits all four human Notch paralogues. CHO-K1 cells were transfected with RBPJ{kappa}-Luc and pRL-CMV. Notch signalling was initiated by expressing active forms of each human Notch paralogue ({Delta}N-hN1-4) in the presence or absence of mDvl2. Data are presented as mean fold change (±s.e.m.) in RLU compared with each Notch construct alone. Dvl2 inhibited each Notch paralogue (***P<0.001, one-way ANOVA and Tukey's post-hoc test, n=3). (B) mDvl2 does not inhibit Notch cleavage. Cells transfected with RBPJ{kappa}-lacZ and pRL-CMV were also transfected with vectors encoding {Delta}N-mN1 or mDvl2. Lysates were analysed by immunoblotting to examine β-gal reporter gene activity and {Delta}N-mN1 cleavage (NICD Val1744). R. luciferase is a loading control. (C,D) NICD released from {Delta}N-mN1 translocates to the nucleus even in the presence of mDvl2. (C) Cells expressing {Delta}N-mN1-GFP and mDvl2-V5, as indicated, were fixed and immunostained for GFP (green) and V5 (red) epitopes. DAPT treatment to inhibit {gamma}-secretase function prevented nuclear translocation of NICD. (D) CHO-K1 cells expressing {Delta}N-mN1 and mDvl2, as indicated, were fractionated and nuclear accumulation of NICD was analysed by immunoblotting the nuclear fraction (Nuc) and total lysates (Total). LaminB1 is a loading control. Positions of molecular weight markers (in kDa) are indicated.

 

Figure 4
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  		Fig. 4. Dishevelled inhibits RBPJ{kappa} downstream of Notch. (A-D) CHO-K1 cells were transfected with RBPJ{kappa}-Luc (A,C,D) or NRE Su(H)-Luc (B) and pRL-CMV. Notch and Wnt signalling were activated by co-expression of {Delta}N-mN1 or an active form of RBPJ{kappa}, VP16-RBPJ{kappa} and mDvl2 (A), XSu(H)-ANK and XDvl2 (B), or VP16-RBPJ{kappa}, Su(H)-VP16, mDvl2 and Drosophila Dsh (C,D), as indicated. Data are presented as mean fold change (±s.e.m.) in RLU relative to each Notch pathway component alone. (A) mDvl2 inhibited both {Delta}N-mN1 and VP16-RBPJ{kappa} similarly. (B) XDvl2 inhibits XSu(H)-ANK. (C,D) Drosophila Dsh and mDvl2 inhibited VP16-RBPJ{kappa} (C) and Su(H)-VP16 (D). ***P<0.001, one-way ANOVA and Tukey's post-hoc test, n≥3.

 

Figure 5
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  		Fig. 5. Dishevelled binds RBPJ{kappa} within the nucleus and reduces RBPJ{kappa} activity. (A-C) Dishevelled binds CSL proteins even in the absence of NICD. CHO-K1 cells expressing mDvl2-myc and RBPJ{kappa}-GFP (A,C) or XSu(H)-ANK-myc and XDvl2-GFP (B) were subjected to immunoprecipitation using GFP-Trap beads. Immunoprecipitation samples were analysed by immunoblotting for myc and GFP, alongside total lysates. (C) Cells were treated with 5 μM DAPT to prevent the generation of NICD by cleavage of the endogenous Notch protein (see Fig. 3C). (D) Dishevelled binds RBPJ{kappa} within the nucleus. The soluble nuclear fraction was isolated from CHO-K1 cells and subjected to immunoprecipitation with Dvl2 antibody or control IgG. Immunoprecipitation samples and nuclear input (Nuc) were analysed by immunoblotting with RBPJ{kappa} and Dvl2 antibodies (indicated by arrows; asterisk indicates a non-specific band). (E-G) Wnt signalling reduces the amount of RBPJ{kappa} found within the active transcription factor pool. Cells expressing Wnt1, mDvl2 and VP16-RBPJ{kappa}, as indicated, were fractionated to enrich for active transcription factors and subjected to immunoblotting with VP16 and RBPJ{kappa} antibodies. LaminB1 is a loading control. Wnt1 (E) and mDvl2 (F) reduce the amount of VP16-RBPJ{kappa} within the fraction containing active transcription factors. Wnt1 reduces the level of endogenous RBPJ{kappa} within the same fraction (G). Positions of molecular weight markers (in kDa) are shown.

 

Figure 6
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  		Fig. 6. Dishevelled DIX and PDZ domains are required for inhibition of RBPJ{kappa}. (A) Schematic of the structure of Dishevelled and the deletion constructs used. (B) The C terminus of mDvl2 is not required for inhibition of RBPJ{kappa} activity. CHO-K1 cells were transfected with RBPJ{kappa}-Luc and pRL-CMV, and vectors encoding {Delta}N-mN1 and the mDvl2 constructs illustrated. Data are presented as mean fold change (±s.e.m.) in RLU relative to {Delta}N-mN1 alone. Both {Delta}C-mDvl2 and mDvl2, but not {Delta}N-mDvl2, inhibit Notch signalling (one-way ANOVA and Tukey's post-hoc test, n≥3). (C-E) XDvl2 but not Ds1 inhibits endogenous Notch signalling in vivo. Xenopus laevis embryos were injected in one blastomere of a two-cell embryo with mRNA encoding β-gal and XDvl2 (C,C') or β-gal and Ds1 (D,D'). Ciliated cell precursors were detected by α-tubulin expression and the injected side was determined by X-Gal staining. Images of the uninjected (UI) and injected sides of the same embryo are shown. (E) Ciliated cell precursors were quantified as in Fig. 2D. Data are presented as the mean number of precursors counted (±s.e.m.). XDvl2 significantly increased ciliated cell precursor specification but Ds1 did not (two-tailed paired t-test). XDvl2 also increased precursor specification compared with Ds1 (two-way ANOVA and Bonferroni's post-hoc test). (F) Ds1 does not inhibit XSu(H) activity. CHO-K1 cells were transfected with NRE Su(H)-Luc and pRL-CMV, and vectors encoding XSu(H)-ANK, XDvl2 or Ds1. Data are presented as mean fold change (±s.e.m.) in RLU relative to XSu(H)-ANK alone (one-way ANOVA and Tukey's post-hoc test, n=3). (G) Ds1 shows greatly reduced XSu(H) binding. CHO-K1 cells expressing XSu(H)-ANK-myc and XDvl2-GFP or Ds1-GFP were subjected to immunoprecipitation using GFP-Trap beads. Immunoprecipitation samples were analysed by immunoblotting for myc and GFP, alongside total lysates. Positions of molecular weight markers (in kDa) are shown. Scale bar: 500 μm. **P<0.01; ***P<0.001; ns, P>0.05.

 

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